Biomedical engineering faces many challenges in the development of tissue that can be used to facilitate healing. For example, a “replacement” tissue should promote tissue regeneration. In doing so, the new tissue must be compatible with the recipient tissue so that neighboring cells accept the replacement. Importantly, the replacement tissue should overcome (or avoid) the immunologic responses typically elicited by the addition of a “foreign body” to a biological system.
Furthermore, the replacement tissue should exhibit the properties and function of the tissue that it is replacing. For example, the replacement tissue should exhibit similar mechanical and structural properties of the original tissue or, at a minimum, not interfere with the native environment. The replacement tissue may act as a scaffold and/or retain biological properties to promote cellular regeneration. Finally, the replacement tissue should not stimulate scar formation that limits tissue regeneration or inhibits the natural function of the underlying tissue.
One particularly challenging embodiment of tissue engineering is the production of nerve grafts that can be used to help regeneration of severed nerves. In direct nerve repair, when the ends of a severed nerve are reconnected (without an interpositional graft), axons will attempt to regrow from the proximal nerve into the distal nerve. In this context, following nerve injury, the distal nerve undergoes a process known as Wallerian degeneration, which involves the breakdown and clearance of nerve elements including the nonfunctional distal axons and their myelin sheaths. In part because of this process, axonal and myelin debris have long been believed to have growth-inhibitory effects that curtail nerve regeneration and may be a mechanical barrier to axonal growth.
Extensive evidence indicates that nerve regeneration is slower when the process of Wallerian degeneration is delayed. Accordingly, it has been widely accepted that the clearance of nerve elements improves axonal growth in the distal nerve. This premise has been extrapolated to nerve grafting and has fostered the belief that cellular debris must also be removed from nerve grafts in order to promote axonal growth. Removal of residual cellular material has also been thought to be necessary to minimize pathogens in the tissue and eliminate immunogenic material that might lead to graft immunorejection. Consequently, processing methods used for nerve grafts have routinely involved rigorous decellularization techniques, even at the expense of disrupting extracellular matrix (ECM) structures and nerve graft integrity that support the regenerative process.
Nerve damage often results in the loss of clean ends for direct repair or a gap created by nerve tissue damage. In this case nerve repair requires the an interpositional graft to bridge the deficit and restore nerve continuity.
For instance, U.S. Pat. No. 7,402,319 (which is incorporated herein in its entirety) describes an acellular nerve allograft that can reinstate nerve continuity and, under the right circumstances, can lead to nerve regeneration. This type of nerve graft is obtained by soaking nerve tissue in several series of solutions with sulfobetaines and anionic surface-active detergents (e.g., Triton X-200), which are used to decellularize the nerve tissue.
Hudson et al. applied a decellularization technique containing Triton X-200™, sulfobetaine-16, and sulfobetaine-10 on rat nerves and reported very high levels of extraction. (Hudson T W, Liu S Y, Schmidt C E. Tissue Eng. 10: 1346-58, 2004). Rodent nerves, however, contain a single nerve bundle (fascicle) and the outer nerve sheaths of rodent nerves have little resemblance to the nerves of larger animals. Most nerves of larger animals (e.g., rabbit, human) have multiple nerve bundles embedded in a collagenous inner epineurium (connective tissue surrounding the nerve bundles). The sheath and intrafascicular structures of human nerves are expansive and have a profound influence on nerve integrity and permeability, including properties that impact tissue extraction. Therefore, knowledge about decellularization and extraction of rodent nerve is at best suggestive of the extraction expected when applied to the nerves from larger animals.
In summary, there remains a need to improve tissue replacements, particularly in the context of nerve tissue. The improved tissue replacement should maintain native structural and bioactive characteristics of the tissue it is replacing, including, for example, laminin activity, and be able to incorporate bioactive compounds or molecules where necessary to promote rapid regeneration, and stimulate tissue repair and regeneration without scarring that can reduce tissue mobility and integrity.